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- PDB-7tb3: cryo-EM structure of MBP-KIX-apoferritin -

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Entry
Database: PDB / ID: 7tb3
Titlecryo-EM structure of MBP-KIX-apoferritin
ComponentsIsoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
KeywordsSTRUCTURAL PROTEIN / Protein Engineering / Simulation / Peptide Therapeutics / Acute Myeloid Leukemia
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / peptide lactyltransferase (CoA-dependent) activity / NFE2L2 regulating ER-stress associated genes / NFE2L2 regulating inflammation associated genes / Activation of the TFAP2 (AP-2) family of transcription factors / histone H3K18 acetyltransferase activity / N-terminal peptidyl-lysine acetylation / histone H3K27 acetyltransferase activity / LRR FLII-interacting protein 1 (LRRFIP1) activates type I IFN production ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / peptide lactyltransferase (CoA-dependent) activity / NFE2L2 regulating ER-stress associated genes / NFE2L2 regulating inflammation associated genes / Activation of the TFAP2 (AP-2) family of transcription factors / histone H3K18 acetyltransferase activity / N-terminal peptidyl-lysine acetylation / histone H3K27 acetyltransferase activity / LRR FLII-interacting protein 1 (LRRFIP1) activates type I IFN production / NFE2L2 regulates pentose phosphate pathway genes / regulation of smoothened signaling pathway / NFE2L2 regulating MDR associated enzymes / MRF binding / RUNX1 regulates transcription of genes involved in differentiation of myeloid cells / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / Regulation of FOXO transcriptional activity by acetylation / RUNX3 regulates NOTCH signaling / NOTCH4 Intracellular Domain Regulates Transcription / Nuclear events mediated by NFE2L2 / Regulation of NFE2L2 gene expression / Regulation of gene expression by Hypoxia-inducible Factor / negative regulation of transcription by RNA polymerase I / NOTCH3 Intracellular Domain Regulates Transcription / TRAF6 mediated IRF7 activation / NFE2L2 regulating tumorigenic genes / NFE2L2 regulating anti-oxidant/detoxification enzymes / embryonic digit morphogenesis / protein-lysine-acetyltransferase activity / negative regulation of ferroptosis / protein acetylation / homeostatic process / Notch-HLH transcription pathway / Formation of paraxial mesoderm / ferroxidase / positive regulation of transforming growth factor beta receptor signaling pathway / acetyltransferase activity / FOXO-mediated transcription of cell death genes / stimulatory C-type lectin receptor signaling pathway / Zygotic genome activation (ZGA) / TP53 Regulates Transcription of Genes Involved in Cytochrome C Release / autolysosome / ferroxidase activity / histone acetyltransferase complex / canonical NF-kappaB signal transduction / Attenuation phase / histone acetyltransferase activity / cellular response to nutrient levels / histone acetyltransferase / positive regulation of double-strand break repair via homologous recombination / regulation of cellular response to heat / : / Regulation of lipid metabolism by PPARalpha / NPAS4 regulates expression of target genes / Neutrophil degranulation / endocytic vesicle lumen / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / Transcriptional and post-translational regulation of MITF-M expression and activity / ferric iron binding / CD209 (DC-SIGN) signaling / BMAL1:CLOCK,NPAS2 activates circadian expression / autophagosome / SUMOylation of transcription cofactors / Activation of gene expression by SREBF (SREBP) / iron ion transport / Heme signaling / Transcriptional activation of mitochondrial biogenesis / Formation of the beta-catenin:TCF transactivating complex / PPARA activates gene expression / Cytoprotection by HMOX1 / protein destabilization / chromatin DNA binding / Evasion by RSV of host interferon responses / Transcriptional regulation of white adipocyte differentiation / NOTCH1 Intracellular Domain Regulates Transcription / positive regulation of protein localization to nucleus / Pre-NOTCH Transcription and Translation / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / transcription coactivator binding / tau protein binding / Activation of anterior HOX genes in hindbrain development during early embryogenesis / p53 binding / cellular response to UV / : / transcription corepressor activity / rhythmic process / HATs acetylate histones / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / TRAF3-dependent IRF activation pathway / protein-containing complex assembly / transcription regulator complex / Estrogen-dependent gene expression / DNA-binding transcription factor binding / damaged DNA binding / RNA polymerase II-specific DNA-binding transcription factor binding / intracellular iron ion homeostasis / transcription coactivator activity / response to hypoxia / immune response
Similarity search - Function
Coactivator CBP, KIX domain / Nuclear receptor coactivator, CREB-bp-like, interlocking / Nuclear receptor coactivator, CREB-bp-like, interlocking domain superfamily / Creb binding / Zinc finger, TAZ-type / TAZ domain superfamily / TAZ zinc finger / Zinc finger TAZ-type profile. / TAZ zinc finger, present in p300 and CBP / : ...Coactivator CBP, KIX domain / Nuclear receptor coactivator, CREB-bp-like, interlocking / Nuclear receptor coactivator, CREB-bp-like, interlocking domain superfamily / Creb binding / Zinc finger, TAZ-type / TAZ domain superfamily / TAZ zinc finger / Zinc finger TAZ-type profile. / TAZ zinc finger, present in p300 and CBP / : / Histone acetyltransferase p300-like, PHD domain / Coactivator CBP, KIX domain / CREB-binding protein/p300, atypical RING domain / CBP/p300-type histone acetyltransferase domain / CBP/p300, atypical RING domain superfamily / KIX domain / CREB-binding protein/p300, atypical RING domain / KIX domain profile. / CBP/p300-type histone acetyltransferase (HAT) domain profile. / Histone acetyltransferase Rtt109/CBP / Histone acetylation protein / Histone acetylation protein / Coactivator CBP, KIX domain superfamily / Serum Albumin; Chain A, Domain 1 / Zinc finger ZZ-type signature. / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Nuclear receptor coactivator, interlocking / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / Zinc finger, RING/FYVE/PHD-type / Up-down Bundle / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Ferritin heavy chain / CREB-binding protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsZhang, K. / Horikoshi, N. / Li, S. / Powers, A. / Hameedi, M. / Pintilie, G. / Chae, H. / Khan, Y. / Suomivuori, C. / Dror, R. ...Zhang, K. / Horikoshi, N. / Li, S. / Powers, A. / Hameedi, M. / Pintilie, G. / Chae, H. / Khan, Y. / Suomivuori, C. / Dror, R. / Sakamoto, K. / Chiu, W. / Wakatsuki, S.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: ACS Cent Sci / Year: 2022
Title: Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia.
Authors: Kaiming Zhang / Naoki Horikoshi / Shanshan Li / Alexander S Powers / Mikhail A Hameedi / Grigore D Pintilie / Hee-Don Chae / Yousuf A Khan / Carl-Mikael Suomivuori / Ron O Dror / Kathleen M ...Authors: Kaiming Zhang / Naoki Horikoshi / Shanshan Li / Alexander S Powers / Mikhail A Hameedi / Grigore D Pintilie / Hee-Don Chae / Yousuf A Khan / Carl-Mikael Suomivuori / Ron O Dror / Kathleen M Sakamoto / Wah Chiu / Soichi Wakatsuki /
Abstract: Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures ...Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 Å resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.
History
DepositionDec 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Assembly

Deposited unit
A: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
B: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
C: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
D: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
E: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
F: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
G: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
H: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
I: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
J: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
K: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
L: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
M: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
N: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
O: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
P: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
Q: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
R: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
S: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
T: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
U: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
V: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
W: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
X: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed


Theoretical massNumber of molelcules
Total (without water)729,80324
Polymers729,80324
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed / Histone lysine acetyltransferase CREBBP / Protein-lysine acetyltransferase CREBBP


Mass: 30408.438 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CREBBP, CBP, Fth1, Fth
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q92793, UniProt: P09528, histone acetyltransferase, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MBP-KIX-apoferritin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.7 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MBP-KIX-apoferritin
Specimen supportGrid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 43.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1915
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2EPU2.7image acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.41model fitting
12cryoSPARC23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 167662
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37386 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Target criteria: Q-score

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